Apparatus, system and method for collecting a target material

ABSTRACT

This disclosure is directed to an apparatus, system and method for retrieving a target material from a sample. An enrichment agent may be added to a vessel that contains the sample for positive selection, or, in other words, to select or aid in selecting the target material from amongst the remainder of the sample. The enrichment agent may be, for example, immunomagnetic beads, buoyant beads, high-density beads, chemicals to change the density of the target material, or the like.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.15/173,692, filed Jun. 5, 2016, which is a continuation of applicationSer. No. 15/019,697, filed Feb. 9, 2016, which is a continuation-in-partof application Ser. No. 14/883,071, filed Oct. 14, 2015, which claimsthe benefit of Provisional Application No. 62/068,480, filed Oct. 24,2014, and which is a continuation-in-part of application Ser. No.14/665,368, filed Mar. 23, 2015, (now U.S. Pat. No. 9,217,697, issuedDec. 22, 2015), which is a continuation-in-part of application Ser. No.14/610,522, filed Jan. 30, 2015, which claims the benefit of ProvisionalApplication No. 61/935,457, filed Feb. 4, 2014, and which is acontinuation-in-part of application Ser. No. 14/495,445, filed Sep. 24,2014, (now U.S. Pat. No. 9,039,999, issued May 26, 2015), which is acontinuation-in-part of application Ser. No. 14/090,337, filed Nov. 26,2013, which claims the benefit(s) of Provisional Application No.61/732,029, filed Nov. 30, 2012, of Provisional Application No.61/745,094, filed Dec. 21, 2012, of Provisional Application No.61/791,883, filed Mar. 15, 2013, of Provisional Application No.61/818,301, filed May 1, 2013, and of Provisional Application No.61/869,866, filed Aug. 26, 2013, and which is a continuation-in-part ofapplication Ser. No. 14/266,939, filed May 1, 2014, (now expired) whichclaims the benefit(s) of Provisional Application No. 61/818,301, filedMay 1, 2013, of Provisional Application No. 61/869,866, filed Aug. 26,2013, and of Provisional Application No. 61/935,457, filed Feb. 4, 2014.

TECHNICAL FIELD

This disclosure relates generally to density-based fluid separation and,in particular, to retrieving a target material from a suspension.

BACKGROUND

Suspensions often include materials of interests that are difficult todetect, extract and isolate for analysis. For instance, whole blood is asuspension of materials in a fluid. The materials include billions ofred and white blood cells and platelets in a proteinaceous fluid calledplasma. Whole blood is routinely examined for the presence of abnormalorganisms or cells, such as ova, fetal cells, endothelial cells,parasites, bacteria, and inflammatory cells, and viruses, including HIV,cytomegalovirus, hepatitis C virus, and Epstein-Barr virus. Currently,practitioners, researchers, and those working with blood samples try toseparate, isolate, and extract certain components of a peripheral bloodsample for examination. Typical techniques used to analyze a bloodsample include the steps of smearing a film of blood on a slide andstaining the film in a way that enables certain components to beexamined by bright field microscopy.

On the other hand, materials of interest that occur in a suspension withvery low concentrations are especially difficult if not impossible todetect and analyze using many existing techniques. Consider, forinstance, circulating tumor cells (“CTCs”), which are cancer cells thathave detached from a tumor, circulate in the bloodstream, and may beregarded as seeds for subsequent growth of additional tumors (i.e.,metastasis) in different tissues. The ability to accurately detect andanalyze CTCs is of particular interest to oncologists and cancerresearchers. However, CTCs occur in very low numbers in peripheral wholeblood samples. For instance, a 7.5 ml sample of peripheral whole bloodsample that contains as few as 5 CTCs is considered clinically relevantfor the diagnosis and treatment of a cancer patient. In other words,detecting 5 CTCs in a 7.5 ml blood sample is equivalent to detecting 1CTC in a background of about 40-60 billion red and white blood cells,which is extremely time consuming, costly and difficult to accomplishusing blood film analysis.

As a result, practitioners, researchers, and those working withsuspensions continue to seek systems and methods for accurate analysisof suspensions for the presence or absence rare materials of interest.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show an example collector.

FIGS. 2A-2B show an example collector.

FIGS. 2C-2D show an example collector.

FIGS. 3A-3C show example collectors.

FIGS. 4A-4B show an example collector-processing vessel system.

FIG. 4C shows an example collector-processing vessel system.

FIGS. 5A-5B show an example collector-canopy system.

FIGS. 6A-6G show example sealing rings.

FIG. 7 shows a flow diagram of an example method for retrieving a targetmaterial.

FIGS. 8A-8D show example float and tube systems.

FIG. 9 shows a sample having undergone density-based separation.

FIGS. 10A-10B show a seal being formed by a clamp.

FIGS. 11A-11F show an example system retrieving a target material.

FIG. 12 shows a flow diagram of an example method for retrieving atarget material.

DETAILED DESCRIPTION

This disclosure is directed to an apparatus, system and method forretrieving a target material from a sample. An enrichment agent may beadded to a vessel that contains the sample for positive selection, or,in other words, to select or aid in selecting the target material fromamongst the remainder of the sample. The enrichment agent may be, forexample, immunomagnetic beads, buoyant beads, high-density beads,chemicals to change the density of the target material, or the like.

Collector

FIG. 1A shows an isometric view of a collector 100. FIG. 1B shows across-sectional view of the collector 100 taken along the line I-I shownin FIG. 1A. Dot-dashed line 102 represents the central orhighest-symmetry axis of the collector 100. The collector 100 may besized and shaped to fit within a primary vessel containing or capable ofholding a suspension, the suspension suspected of including a targetmaterial. The collector 100 funnels the target material from thesuspension through a cannula 106 and into a processing vessel (notshown) to be located within a cavity 108. The collector 100 includes themain body 104 which includes a first end 110 and a second end 112. Aseal may be formed between the second end 112 and an inner wall of theprimary vessel to maintain a fluid-tight sealing engagement before,during, and after centrifugation and to inhibit any portion of thesuspension from being located or flowing between an inner wall of theprimary vessel and a main body 104 of the collector 100. The seal may beformed by an interference fit, a grease (such as vacuum grease), anadhesive, an epoxy, by bonding (such as by thermal bonding), by welding(such as by ultrasonic welding), by clamping (such as with a ring orclamp), an insert (such as an O-ring or a collar) that fits between thesecond end 112 and the inner wall of the primary vessel, or the like.The main body 104 may be any appropriate shape, including, but notlimited to, cylindrical, triangular, square, rectangular, or the like.The collector 100 also includes an internal funnel 114 which is aconcave opening. The funnel 114 may taper toward the cannula 106 fromthe second end 112. The funnel 114 channels a target material from belowthe second end 112 into the cannula 106 which is connected to, and influid communication with, an apex of the funnel 114. The apex of thefunnel 114 has a smaller diameter than the mouth of the funnel 114. Thefunnel 114 is formed by a tapered wall that may be straight,curvilinear, arcuate, or the like. The funnel 114 may be any appropriateshape, including, but not limited to, tubular, spherical, domed,conical, rectangular, pyramidal, or the like. Furthermore, the outermostdiameter or edge of the funnel 114 may be in continuous communication orconstant contact (i.e. sit flush) with the inner wall of the primaryvessel such that no dead space is present between the second end 112 ofthe collector 100 and the inner wall of the primary vessel.

The cannula 106, such as a tube or a needle, including, but not limitedto a non-coring needle, extends from the apex of the funnel 114 and intothe cavity 108. In the example of FIG. 1, the cavity 108 is a concaveopening extending from the first end 110 into the main body 104 and mayaccept and support the processing vessel (not shown). The cavity 108 maybe any appropriate depth to accept and support the processing vessel(not shown). The cannula 106 may extend any appropriate distance intothe cavity 108 in order to puncture the base of, or be inserted into,the processing vessel (not shown). The cannula 106 may include a flattip, a beveled tip, a sharpened tip, or a tapered tip. Furthermore, thecavity 108 may be any appropriate shape, including, but not limited to,tubular, spherical, domed, conical, rectangular, pyramidal, or the like.The cavity 108 may be threaded to engage a threaded portion of theprocessing vessel (not shown).

The collector 100 may also include a retainer (not shown) to prevent thecollector 100 from sliding relative to the primary vessel, therebykeeping the collector 100 at a pre-determined height within the primaryvessel. The retainer (not shown) may be a shoulder extending radiallyfrom the first end 110, a clip, a circular protrusion that extendsbeyond the circumference of the cylindrical main body 104, a detent, orthe like.

FIG. 2A shows an isometric view of a collector 200. FIG. 2B shows across-section view of the collector 200 taken along the line II-II shownin FIG. 2A. Dot-dashed line 202 represents the central orhighest-symmetry axis of the collector 200. The collector 200 is similarto the collector 100, except that the collector 200 includes a main body204 that is more elongated than the main body 104 of the collector 100in order to accommodate a greater portion of the processing vessel (notshown). The main body 204 includes a first end 206 and a second end 208.A seal may be formed between the second end 208 and an inner wall of theprimary vessel to maintain a fluid-tight sealing engagement before,during, and after centrifugation and to inhibit any portion of thesuspension flowing between an inner wall of the primary vessel and themain body 204 of the collector 200. The seal may be formed by aninterference fit, a grease (such as vacuum grease), an adhesive, anepoxy, by bonding (such as thermal bonding), by welding (such asultrasonic welding), clamping (such as with a ring or clamp), an insert(such as an O-ring or a collar) that fits between the second end 208 andthe inner wall of the primary vessel, or the like.

The first end 206 includes a cavity 212 dimensioned to accept and holdat least a portion of the processing vessel (not shown). The cavity 212may have a tapered or stepped bottom end 220 on which the processingvessel (not shown) may rest. The first end 206 may also include at leastone cut-out 210 to permit proper grip of the processing vessel (notshown) for insertion and removal. The collector 200 funnels the targetmaterial from the suspension into an internal funnel 222 at the secondend 208, through a cannula 214, and into a processing vessel (not shown)located within the cavity 212. The cannula 214 may rest on a shelf 224so that an inner bore of the cannula 214 sits flush with an inner wallof the funnel 222, as shown in FIG. 2B.

The collector 200 may include a shoulder 216, which extendscircumferentially around the main body 204. The shoulder 216 may belarger than the inner diameter of the primary vessel so as to rest onthe open end of the primary vessel and, upon applying a lock ring (notshown) to the outside of the primary vessel and the shoulder 216, toinhibit movement of the collector 200 relative to the primary vessel.The lock ring (not shown) applies pressure to the primary vessel alongthe shoulder 216. The lock ring may be a two-piece ring, a one piecering wrapping around the full circumference of the primary vessel, or aone piece ring wrapping around less than the full circumference of theprimary vessel, such as one-half (½), five-eighths (⅝), two-thirds (⅔),three-quarters (¾), seven-eighths (⅞), or the like. Alternatively, theshoulder 216 may fit within the primary vessel. Alternatively, theshoulder 216 may be a clip, such that the shoulder 216 may include acatch into which the primary vessel may be inserted to inhibit movementof the collector 200 relative to the primary vessel. Alternatively, theshoulder 216 may form an interference fit with the inner wall of theprimary vessel around which a seal ring may be placed.

As shown in FIG. 2A, the collector 200 may include at least one window218 to access the cavity 212 through an inner wall of the main body 204.The at least one window 218 permits an operator to confirm properplacement of the processing vessel (not shown) within the cavity 212.The at least one window 218 also allows fluid discharged from thecannula 214 to flow out of the collector 200 and into a space formedbetween the collector 200 and the primary vessel (not shown) and abovethe seal between the second end 208 and the inner wall of the primaryvessel.

FIG. 2C shows an isometric view of a collector 230. FIG. 2D shows across-section view of the collector 230 taken along the line shown inFIG. 2C. The collector 230 is similar to the collector 200, except thatthe collector 230 includes a main body 238 including an extension 234extending away from a first end 232 and a lid 236 to at leasttemporarily seal an opening 240 within the extension 234. The opening240 may be in fluid communication with the cavity 212 at the first end232. The lid 236 may removable, puncturable and resealable (e.g. a flaplid), or puncturable and non-resealable (e.g. a foil lid). The extension234 may be sized to accept the lid 236 when punctured such that aportion of the lid 236 does not extend into the cavity 212 at the firstend 232. Note that the collector 230 does not include the at least onecut-out 210.

FIG. 3A shows an isometric view of a collector 300. Dot-dashed line 302represents the central or highest-symmetry axis of the collector 300.The collector 300 includes a first side 304, a second side 306, and abore 308. An inner wall 312 to form the bore 308, 324, and 334 may betapered (i.e. becoming narrower from the first side 304 to the secondside 306; or becoming wider from the first side 304 to the second 306),as shown in FIG. 3A.

The first and second sides 304 and 306 may be connected to the innerwall 312 via straight walls (i.e. first and second sides 304 and 306 areplanar), tapered walls, or at least partially arcuate walls.

The collector 300 may be sized and shaped to fit within a vesselcontaining or capable of holding a suspension. The collector 300 fitsagainst an inner wall of the vessel, such that no portion of thesuspension may be located between the inner wall of the vessel and themain body 310 of the collector 300. The collector 300 gathers a samplewithin the bore 308. The bore 308 may be expandable, such that thediameter of the bore 308 may increase during centrifugation and thenreturn to a resting diameter when not under centrifugation. Expandingthe diameter may allow for less constricted flow of fluid and suspensioncomponents during centrifugation. The collector 300 may be composed of aceramic, metal, polymer, flexible polymer, glass, organic or inorganicmaterials, or the like.

FIG. 3B shows an isometric view of a collector 320. The collector 320 issimilar to the collector 300, except that an inner wall 322 of thecollector 320 may be straight (i.e. having a uniform diameter from thefirst side 304 to the second side 306). FIG. 3C shows an isometric viewof a collector 330. The collector 330 is similar to the collector 300,except that an inner wall 332 of the collector 330 may be at leastpartially arcuate (i.e. concave, convex, or curvilinear).

The collector may also include a filter (not shown). The filter (notshown) may be located at the second side or in the bore. The filter (notshown) is configured to provide a more pure sample by permitting atarget material to pass through, while inhibiting non-target materialfrom passing through.

The main body can be composed of a variety of different materialsincluding, but not limited to, a ceramic; metals; organic or inorganicmaterials; and plastic materials, such as polyoxymethylene (“Delrin®”),polystyrene, acrylonitrile butadiene styrene (“ABS”) copolymers,aromatic polycarbonates, aromatic polyesters, carboxymethylcellulose,ethyl cellulose, ethylene vinyl acetate copolymers, nylon, polyacetals,polyacetates, polyacrylonitrile and other nitrile resins,polyacrylonitrile-vinyl chloride copolymer, polyamides, aromaticpolyamides (“aramids”), polyamide-imide, polyarylates, polyaryleneoxides, polyarylene sulfides, polyarylsulfones, polybenzimidazole,polybutylene terephthalate, polycarbonates, polyester, polyester imides,polyether sulfones, polyetherimides, polyetherketones,polyetheretherketones, polyethylene terephthalate, polyimides,polymethacrylate, polyolefins (e.g., polyethylene, polypropylene),polyallomers, polyoxadiazole, polyparaxylene, polyphenylene oxides(PPO), modified PPOs, polystyrene, polysulfone, fluorine containingpolymer such as polytetrafluoroethylene, polyurethane, polyvinylacetate, polyvinyl alcohol, polyvinyl halides such as polyvinylchloride, polyvinyl chloride-vinyl acetate copolymer, polyvinylpyrrolidone, polyvinylidene chloride, specialty polymers, polystyrene,polycarbonate, polypropylene, acrylonitrite butadiene-styrene copolymer,butyl rubber, ethylene propylene diene monomer; and combinationsthereof.

The cannula can be composed of a variety of different materialsincluding, but not limited to, a ceramic; metals; organic or inorganicmaterials; and plastic materials, such as a polypropylene, acrylic,polycarbonate, or the like; and combinations thereof. The cannula mayhave a tip along a longitudinal axis of the cannula.

Collector-Processing Vessel System

FIG. 4A shows an exploded view of the example collector 200 andprocessing vessel 402. FIG. 4B shows a cross-sectional view of theprocessing vessel 402 inserted into the cavity 212 at the first end 206of the collector 200 taken along the line IV-IV shown in FIG. 4A. Thecollector 200 and processing vessel 402 form a collector-processingvessel system 400. The processing vessel 402 may be an Eppendorf tube, asyringe, or a test tube and has a closed end 404 and an open end 406.The open end 406 is sized to receive a cap 408. The cap 408 may becomposed of re-sealable rubber or other suitable re-sealable materialthat can be repeatedly punctured with a needle or other sharp implementto access the contents stored in the processing vessel 402 interior andre-seals when the needle or implement is removed. Alternatively, theprocessing vessel 402 may also have two open ends that are sized toreceive caps. The processing vessel 402 may have a tapered geometry thatwidens or narrows toward the open end 406; the processing vessel 402 mayhave a generally cylindrical geometry; or, the processing vessel 402 mayhave a generally cylindrical geometry in a first segment and acone-shaped geometry in a second segment, where the first and secondsegments are connected and continuous with each other. Although at leastone segment of the processing vessel 402 has a circular cross-section,in other embodiments, the at least one segment can have elliptical,square, triangular, rectangular, octagonal, or any other suitablecross-sectional shape. The processing vessel 402 can be composed of atransparent, semitransparent, opaque, or translucent material, such asplastic or another suitable material. The processing vessel includes acentral axis 414, which when inserted into the cavity 212 is coaxialwith the central axis 202 of the collector 200. The processing vessel402 may also include a plug 410 at the closed end 404 to permit theintroduction of the target material or to exchange or replace the targetmaterial with a collection fluid 412. The closed end 404 may be threadedto provide for a threaded connection with a threaded cavity 212 of thecollector 200. The processing vessel 402 may be composed of glass,plastic, or other suitable material.

The plug 410 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access the contents of the processing vessel402 interior or permit introduction of contents into the processingvessel 402 and re-seals when the needle or implement is removed. Theplug 410 may be inserted into the processing vessel 402 such that a sealis maintained between the plug 410 and the processing vessel 402, suchas by an interference fit. Alternatively, the plug 410 can be formed inthe closed end 404 of the processing vessel 402 using heated liquidrubber that can be shaped while warm or hot and hardens as the rubbercools. An adhesive may be used to attach a plug 410 to the inner wall ofthe processing vessel can be a polymer-based adhesive, an epoxy, acontact adhesive or any other suitable material for bonding or creatinga thermal bond. Alternatively, the plug 410 may be injected into theprocessing vessel 402. Alternatively, the plug 410 may be thermallybonded to the processing vessel 402.

In the example of FIG. 4B, the cannula 214 has a tapered tip thatpunctures the plug 410 and extends into an inner cavity of theprocessing vessel 402 with the shaft of the cannula 214 not extendinginto the inner cavity of the processing vessel 402. As explained ingreater detail below, the inner cavity of the processing vessel 402holds the target material. The cannula 214 may be covered by aresealable sleeve (not shown) to prevent the target material fromflowing out unless the processing vessel 402 is in the cavity 212 to adepth that allows the cannula 214 to just penetrate the processingvessel 402. The resealable sleeve (not shown) covers the cannula 214, isspring-resilient, can be penetrated by the cannula 214, and is made ofan elastomeric material capable of withstanding repeated punctures whilestill maintaining a seal.

As shown in FIGS. 4A-4B, the processing vessel 402 may be loaded with acollection fluid 412 prior to insertion into the collector 200. Thecollection fluid 412 displaces the target material, such that when thecollector 200 and processing vessel 402 are inserted into the primaryvessel (not shown) including the target material, and the collector,processing vessel, and primary vessel undergo centrifugation, thecollection fluid 412 flows out of the processing vessel 402 and into theprimary vessel, and, through displacement, such as through buoyantdisplacement (i.e. lifting a material upwards), pushes the targetmaterial through the cannula 214 and into the processing vessel 402.

The collection fluid 412 has a greater density than the density of thetarget material of the suspension (the density may be greater than thedensity of a subset of suspension fractions or all of the suspensionfractions) and is inert with respect to the suspension materials. Thecollection fluid 412 may be miscible or immiscible in the suspensionfluid. Examples of suitable collection fluids include, but are notlimited to, solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), an organic solvent, a liquid wax, anoil, a gas, and combinations thereat olive oil, mineral oil, siliconeoil, immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, and combinations thereof; organic solvents suchas 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol,cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methylether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane,propylene carbonate, tetramethylene sulfones, and ionic liquids;polymer-based solutions; surfactants; perfluoroketones, such asperfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones,hydrofluoroethers, hydrofluorocarbons, perfluorocarbons,perfluoropolyethers, silicon and silicon-based liquids, such asphenylmethyl siloxane; and combinations thereof.

The processing vessel 402 may also include a processing solution (notshown) to effect a transformation on the target material when the targetmaterial enters the processing vessel 402. The processing solution (notshown) may be a preservative, a cell adhesion solution, a dye, or thelike. Unlike the collection fluid 412, most, if not all, of theprocessing solution (not shown) remains within the processing vessel 402upon centrifugation, thereby effecting the transformation on the targetmaterial in one manner or another (i.e. preserving, increasing adhesionproperties, or the like). The processing solution (not shown) may beintroduced as a liquid or as a liquid contained in a casing. The casingmay be dissolvable in an aqueous solution but not in the collectionfluid 412 (such as gel cap); or, the casing may be breakable, such thatthe casing breaks when the processing vessel 402 is shaken in a vortexmixer. Additionally, more than one processing solution may be used.

The processing vessel 402 may include a flexible cap that can be pushedto dispense a pre-determined volume therefrom and onto a substrate, suchas a slide or a well plate. The cap 408 may be flexible or the cap 408may be removed and the flexible cap inserted into the open end 406.Alternatively, the processing vessel 402 may be attached to (i.e. afteraccumulating the target material) or may include a dispenser, which iscapable of dispensing a pre-determined volume of target material fromthe processing vessel 402 onto another substrate, such as a microscopeslide. The dispenser may repeatedly puncture the re-sealable cap 408 orcompress the material within the processing vessel 402 to withdraw anddispense the pre-determined volume of target material onto thesubstrate. Alternatively, the cap 408 may be removed and the dispenser(not shown) may be inserted directly into the processing vessel 402 todispense the buffy coat-processing solution mixture.

FIG. 4C shows an example collection system 420 including the collector300 and a processing vessel 422. The processing vessel 422 is similar tothe processing vessel 402. The processing vessel 422 may be insertedinto the collector 300. The processing vessel 422 may receive the targetmaterial or the portion of the target material. Furthermore, theprocessing vessel 422 may be removed from the collector 300 and thenplaced into another vessel, such as a tube, an Eppendorf tube or aslide, to transfer the target material or the portion of the targetmaterial to the other vessel, such as by centrifugation, for furtherprocessing. Alternatively, an adapter, such as a ferrule, may beincluded to connect the processing vessel 422 to the collector 300. Theadapter may be metal, plastic, glass, or the like.

Collector-Canopy System

FIG. 5A shows an exploded view of the example collector 200 and canopy502. FIG. 5B shows a cross-sectional view of the processing vessel 502inserted into the cavity 212 of the collector 200 taken along the lineV-V shown in FIG. 4A. The collector 200 and canopy 502 form acollector-canopy system 500. The canopy 502 is similar to the processingvessel 402, except that the canopy has a second open end 504. When thecollector-canopy system 500 is inserted into the primary vessel, somefluid within the primary vessel, such as a portion of the suspension, aportion of a suspension fraction, a portion of a clearing fluid, or thelike, may be discharged through the cannula 214. The canopy 502 inhibitsa portion of the fluid in the primary vessel that may be dischargedthrough the cannula 214 from escaping from the opening of the first end206 of the collector 200. The discharged fluid, having been blocked bythe canopy 502, flows out of the second open end 504, and out of thewindow 218. Dashed lines 506 show fluid flow as the fluid is dischargedthrough the cannula 214 and retained by the canopy 502.

Alternatively, when the collector 230 is used, the lid 236 of thecollector 230 inhibits a portion of the fluid in the primary vessel thatmay be discharged through the cannula 214 from escaping from the openingof the first end 206 of the collector 200 in a manner similar to that ofthe canopy 502.

Sealing Ring

FIG. 6A shows an isometric view of a sealing ring 600. FIG. 6B shows atop down view of the sealing ring 600. Dot-dashed line 602 representsthe central or highest-symmetry axis of the sealing ring 600. Thesealing ring 600 includes an inner wall 604, an outer wall 606, and acavity 608. In FIG. 6B, R_(IW) represents the radial distance from thecenter of the sealing ring 600 to the inner wall 604, and R_(OW)represents the radial distance from the center of the sealing ring 600to the outer wall 606. The sealing ring 600 is configured to fit arounda primary vessel, such as a tube. The cavity 608 is sized and shaped toreceive the primary vessel. The sealing ring 600 may be tightened, suchthat the size of the cavity 608 and the radii of the inner and outerwalls 604 and 606 are reduced by circumferentially applying anapproximately uniform, radial force, such as the radial force created bya clamp, around the outer wall 606 directed to the central axis 602 ofthe sealing ring 600. When the sealing ring 600 is tightened around theprimary vessel, the uniform force applied to the sealing ring 600 isapplied to the primary vessel, thereby causing the primary vessel toconstrict. When the radial force is removed from the sealing ring 600,the sealing ring 600 remains tightened and in tension around the primaryvessel.

The sealing ring may be any shape, including, but not limited to,circular, triangular, or polyhedral. FIG. 6C shows an isometric view ofa sealing ring 610. FIG. 6D shows a top down view of the sealing ring610. Sealing ring 610 is similar to sealing ring 600, except sealingring 610 is polyhedral. Dot-dashed line 612 represents the central orhighest-symmetry axis of the sealing ring 610. The sealing ring 610includes an inner wall 614, an outer wall 616, and a cavity 618. Thesealing ring may be composed of a metal, such as brass, a polymer, orcombinations thereof.

Alternatively, as shown in FIG. 6E, a sealing ring 620 may be composedof a piezoelectric material. FIG. 6F shows a top down view of thesealing ring 620. Dot-dashed line 622 represents the central orhighest-symmetry axis of the sealing ring 620. The sealing ring 620 maybe connected to an electric potential source 628, such as a battery, viaa first lead 624 and a second lead 626. The electric potential source628 creates a mechanical strain that causes the sealing ring 620 totighten (i.e. sealing ring 620 radii decrease). The sealing ring 620includes an inner wall 630, an outer wall 632, and a cavity 634. In FIG.6F, R_(IW) represents the radial distance from the center of the sealingring 620 to the inner wall 630, and R_(OW) represents the radialdistance from the center of the sealing ring 620 to the outer wall 632.Alternatively, the sealing ring 620 may be in a naturally tightenedstated. When applying the electric potential the sealing ring 620expands. Alternatively, a portion of the sealing ring may be composed ofthe piezoelectric material, such that the piezoelectric portion acts asan actuator to cause the other portion of the sealing ring to tightenand apply the substantially uniform circumferential pressure on theprimary vessel, thereby constricting the primary vessel to form theseal.

FIG. 6G shows an isometric view of a sealing ring 640. The sealing ringincludes an adjustment mechanism 648 to adjust the inner diameterR_(ID). The collapsible ring includes a processing vessel adapter 642and a primary vessel adapter 646, the first and primary vessel adapters642 and 646 being joined by a band portion 644. The first and primaryvessel adapters 642 and 646 include complementary portions of theadjustment mechanism 648. The adjustment mechanism 648 includes, but isnot limited to, a ratchet, tongue and groove, detents, or the like.

The sealing ring may also include a thermal element, such as a heatedwire. The thermal element may soften the primary vessel forconstriction. Alternatively, the thermal element may melt the primaryvessel to provide a more adherent seal. Alternatively, the thermalelement may cause the sealing ring to compress, thereby forming a sealbetween the primary vessel and float.

Methods

For the sake of convenience, the methods are described with reference toan example suspension of anticoagulated whole blood. But the methodsdescribed below are not intended to be so limited in their scope ofapplication. The methods, in practice, may be used with any kind ofsample, such as a suspension or other biological fluid. For example, asample may be urine, blood, bone marrow, cystic fluid, ascites fluid,stool, semen, cerebrospinal fluid, synovial fluid, nipple aspiratefluid, saliva, amniotic fluid, vaginal secretions, mucus membranesecretions, aqueous humor, vitreous humor, vomit, a suspension derivedfrom a tissue sample or a culture sample, and any other physiologicalfluid or semi-solid. It should also be understood that a target materialmay be a fraction of a sample or a sub-fraction of a fraction, such as aportion of buffy coat. The target material may include an analyte, suchas a cell, such as ova, a nucleated red blood cell, or a circulatingtumor cell (“CTC”), a circulating endothelial cell, a fetal cell, avesicle, a liposome, a protein, a nucleic acid, a biological molecule, anaturally occurring or artificially prepared microscopic unit having anenclosed membrane, a parasite (e.g. spirochetes, such as Borreliaburgdorferi), a microorganism, a virus, or an inflammatory cell; or, thetarget material may be the analytes.

FIG. 7 shows a flow diagram for an example method for retrieving atarget material. In block 702, a sample, such as anticoagulated wholeblood, is obtained. and is added to a primary vessel, such as a testtube. A float may also be added to the primary vessel. For the sake ofconvenience, the methods are described with reference to the float, butthe methods described below are not intended to be so limited in theirapplication and may be performed without the float. However, a depletionagent may be added to the blood prior to or after the blood is added tothe tube and float to remove a sample fraction from the sample or tochange the density of at least a non-target material relative to thedensity of the target material before or after adding the enrichmentagent. For example, the depletion agent may be used to move plateletsaway from the target material, such as by changing the density of theplatelets to be greater than at least the target material, though may beeven greater than or equal to the red blood cells.

Examples of suitable depletion agents include solutions such as, asolution of colloidal silica particles coated with polyvinylpyrrolidone(e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g.OptiPrep), a complex, branch glucan (e.g. Dextran), cesium chloride,sucrose, sugar-based solutions, polymer solutions, multi-phase polymersolutions, tetrameric antibody complexes (e.g. RosetteSep) or the like;or particles, such as beads (composed of at least one of a metal,silica, glass, a polymer, or the like), nanoparticles, metal-basedcompounds, metal complexes, lipids, sugars, or the like. The depletionagent, such as the particles, nanoparticles, complexes, or compounds,may be conjugated to a second complementary molecule, which may bind toa first complementary molecule of at least one antibody. Alternatively,the depletion agent, such as the particles, nanoparticles, complexes, orcompounds, may be conjugated to the at least one antibody, such that thedepletion agent may bind directly to the non-target material. Theparticles may be approximately 0.1-5.0 μm in size.

FIG. 8A shows an isometric view of an example primary vessel and floatsystem 800. The system 800 includes a primary vessel 802 and a float 804suspended within whole blood 806. In the example of FIG. 8A, the primaryvessel 802 has a circular cross-section, a first open end 810, and asecond closed end 808. The open end 810 is sized to receive a cap 812.The primary vessel may also have two open ends that are sized to receivecaps, such as the example tube and separable float system 820 shown FIG.8B. The system 820 is similar to the system 800 except the primaryvessel 802 is replaced by a primary vessel 822 that includes two openends 824 and 826 configured to receive the cap 812 and a cap 828,respectively. The primary vessels 802 and 822 have a generallycylindrical geometry, but may also have a tapered geometry that widens,narrows, or a combination thereof toward the open ends 810 and 824,respectively. Although the primary vessels 802 and 822 have a circularcross-section, in other embodiments, the primary vessels 802 and 822 canhave elliptical, square, triangular, rectangular, octagonal, or anyother suitable cross-sectional shape that substantially extends thelength of the tube. The primary vessels 802 and 822 can be composed of atransparent, semitransparent, opaque, or translucent material, such asplastic or another suitable material. The primary vessels 802 and 822each include a central axis 818 and 830, respectively. The primaryvessel 802 may also include a septum 814, as seen in magnified view 816,at the closed end 808 to permit the removal of a fluid, the suspension,or a suspension fraction, whether with a syringe, a pump, by draining,or the like. The primary vessel 802 may have an inner wall and a firstdiameter.

The septum 814 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access the contents of the primary vessel 802interior and re-seals when the needle or implement is removed. Theseptum 814 may be inserted into the primary vessel 802 such that a sealis maintained between the septum 814 and the primary vessel 802, such asby an interference fit. Alternatively, the septum 814 can be formed inthe openings and/or the bottom interior of the tube using heated liquidrubber that can be shaped while warm or hot and hardens as the rubbercools. An adhesive may be used to attach the septum 814 to the wall ofthe opening and tube interior and can be a polymer-based adhesive, anepoxy, a contact adhesive or any other suitable material for bondingrubber to plastic or creating a thermal bond. Alternatively, the septum814 may be thermally bonded to the primary vessel 802.

The float 804 includes a main body, two teardrop-shaped end caps, andsupport members radially spaced and axially oriented on the main body.Alternatively, the float 804 may not include any support members.Alternatively, the float 804 may include support members which do notengage the inner wall of the primary vessel 802.

In alternative embodiments, the number of support members, supportmember spacing, and support member thickness can each be independentlyvaried. The support members can also be broken or segmented. The mainbody is sized to have an outer diameter that is less than the innerdiameter of the primary vessel 802, thereby defining fluid retentionchannels between the outer surface of the main body and the inner wallof the primary vessel 802. The surfaces of the main body between thesupport members can be flat, curved or have another suitable geometry.The support members and the main body may be a singular structure or maybe separate structures.

Embodiments include other types of geometric shapes for float end caps.The top end cap may be teardrop-shaped, dome-shaped, cone-shaped, or anyother appropriate shape. The bottom end cap may be teardrop-shaped,dome-shaped, cone-shaped, or any other appropriate shape. In otherembodiments, the main body of the float 804 can include a variety ofdifferent support structures for separating samples, supporting the tubewall, or directing the suspension fluid around the float duringcentrifugation. Embodiments are not intended to be limited to theseexamples. The main body may include a number of protrusions that providesupport for the tube. In alternative embodiments, the number and patternof protrusions can be varied. The main body may include a singlecontinuous helical structure or shoulder that spirals around the mainbody creating a helical channel. In other embodiments, the helicalshoulder can be rounded or broken or segmented to allow fluid to flowbetween adjacent turns of the helical shoulder. In various embodiments,the helical shoulder spacing and rib thickness can be independentlyvaried. In another embodiment, the main body may include a supportmember extending radially from and circumferentially around the mainbody. In another embodiment, the support members may be tapered.

The float 804 can be composed of a variety of different materialsincluding, but not limited to, metals; organic or inorganic materials;ferrous plastics; sintered metal; machined metal; plastic materials andcombinations thereof. The primary vessel 802 may have an inner wall anda first diameter. The float 804 can be captured within the primaryvessel 802 by an interference fit, such that under centrifugation, aninner wall of the tube expands to permit axial movement of the float804. When centrifugation stops, the inner wall reduces back to the firstdiameter to induce the interference fit. Alternatively, the inner wallmay not expand and the interference fit may not occur between the float804 and the primary vessel 802, such that the float moves freely withinthe tube before, during, or after centrifugation. The end caps of thefloat may be manufactured as a portion of the main body, thereby beingone singular structure, by machining, injection molding, additivetechniques, or the like; or, the end caps may be connected to the mainbody by a press fit, an adhesive, a screw, any other appropriate methodby which to hold at least two pieces together, or combinations thereof.

The cap 812 may be composed of a variety of different materialsincluding, but not limited to, organic or inorganic materials; plasticmaterials; and combination thereof.

FIG. 8C shows an isometric view of an example primary vessel and floatsystem 840. The system 840 includes a primary vessel 832 and a float 834suspended within whole blood 806. In the example of FIG. 8C, the primaryvessel 832 has a circular cross-section, a first open end 838, and asecond closed end 836. The open end 838 is sized to receive a cap 812.The primary vessel 832 may also have two open ends that are sized toreceive caps. The primary vessel 832 also includes at least one supportmember 842 on an inner wall. The at least one support member 842 mayextend the entire length of the inner wall or a portion thereof. The atleast one support member 842 extends from the inner wall towards acentral axis of the primary vessel 832. The at least one support member842 may extend away from the inner wall approximately 1 to 250 μm. Thesystem 850 is similar to the system 840 except the primary vessel 832 isreplaced by a primary vessel 852 that includes at least one supportmember 854 located in a region of the primary vessel 852 where the float834 is expected to come to rest as a result of centrifugation. Theprimary vessels 832 and 822 have a generally cylindrical geometry, butmay also have a tapered geometry that widens, narrows, or a combinationthereof toward the open ends 838 and 824, respectively. Although theprimary vessels 832 and 822 have a circular cross-section, in otherembodiments, the primary vessels 832 and 822 can have elliptical,square, triangular, rectangular, octagonal, or any other suitablecross-sectional shape that substantially extends the length of the tube.The primary vessels 832 and 822 can be composed of a transparent,semitransparent, opaque, or translucent material, such as plastic oranother suitable material. The primary vessel 832 may also include aseptum 814, at the closed end 836 to permit the removal of a fluid, thesuspension, or a suspension fraction, whether with a syringe, a pump, bydraining, or the like.

In other embodiments, the inner wall of the primary vessel 832 caninclude a variety of different support structures for separatingsamples, supporting the float and/or inner wall, or directing thesuspension fluid around the float during centrifugation. Embodiments arenot intended to be limited to these examples. The inner wall may includea number of protrusions (i.e. bumps) that provide support for the tube.In alternative embodiments, the number and pattern of protrusions can bevaried. The inner wall may include a single continuous helical structureor shoulder that spirals around the inner wall creating a helicalchannel. In other embodiments, the helical shoulder can be rounded orbroken or segmented to allow fluid to flow between adjacent turns of thehelical shoulder. In various embodiments, the helical shoulder spacingand rib thickness can be independently varied. In another embodiment,the main body may include a support member extending radially from andcircumferentially around the inner wall (i.e. raised circular ridges).In another embodiment, the support members may be tapered. Inalternative embodiments, the number of support members, support memberspacing, and support member thickness can each be independently varied.The support members can also be broken or segmented. The support membersand the inner wall may be a singular structure or may be separatestructures.

The septum 814 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access the contents of the primary vessel 832interior and re-seals when the needle or implement is removed. Theseptum 814 may be inserted into the primary vessel 832 such that a sealis maintained between the septum 814 and the primary vessel 832, such asby an interference fit. Alternatively, the septum 814 can be formed inthe openings and/or the bottom interior of the tube using heated liquidrubber that can be shaped while warm or hot and hardens as the rubbercools. An adhesive may be used to attach the septum 814 to the wall ofthe opening and tube interior and can be a polymer-based adhesive, anepoxy, a contact adhesive or any other suitable material for bondingrubber to plastic or creating a thermal bond. Alternatively, the septum814 may be thermally bonded to the primary vessel 832.

The main body of the float 834 may be substantially smooth and may besized to have an outer diameter that is less than the inner diameter ofthe primary vessel 832, thereby defining fluid retention channelsbetween the outer surface of the main body and the inner wall of theprimary vessel 832. The float 834 includes a main body, a dome-shapedtop end cap, and a cone-shaped bottom end cap. Embodiments include othertypes of geometric shapes for float end caps. The top end cap may beteardrop-shaped, dome-shaped, cone-shaped, or any other appropriateshape. The bottom end cap may be teardrop-shaped, dome-shaped,cone-shaped, or any other appropriate shape.

The float 834 can be composed of a variety of different materialsincluding, but not limited to, metals; organic or inorganic materials;ferrous plastics; sintered metal; machined metal; plastic materials andcombinations thereof. The primary vessel 832 may have an inner wall anda first diameter. The float 834 can be captured within the primaryvessel 832 by an interference fit, such that under centrifugation, aninner wall of the tube expands to permit axial movement of the float834. When centrifugation stops, the inner wall reduces back to the firstdiameter to induce the interference fit. Alternatively, the inner wallmay not expand and the interference fit may not occur between the float834 and the primary vessel 832, such that the float moves freely withinthe tube before, during, or after centrifugation. The end caps of thefloat may be manufactured as a portion of the main body, thereby beingone singular structure, by machining, injection molding, additivetechniques, or the like; or, the end caps may be connected to the mainbody by a press fit, an adhesive, a screw, any other appropriate methodby which to hold at least two pieces together, or combinations thereof.

The cap 812 may be composed of a variety of different materialsincluding, but not limited to, organic or inorganic materials; plasticmaterials; and combination thereof.

In block 704, the sample, the float, and the primary vessel undergocentrifugation. FIG. 9 shows an isometric view of the primary vessel andfloat system 800 having undergone density-based separation, such as bycentrifugation. Suppose, for example, the centrifuged whole bloodincludes three fractions. For convenience sake, the three fractionsinclude plasma, buffy coat, and red blood cells. However, when anothersuspension undergoes centrifugation, there may be more than, less than,or the same number of fractions, each fraction having a differentdensity. The suspension undergoes axial separation into three fractionsalong the length the tube based on density, with red blood cells 903located on the bottom, plasma 901 located on top, and buffy coat 902located in between, as shown in FIG. 9. The float 804 may have anyappropriate density to settle within one of the fractions. The densityof the float 804 can be selected so that the float 804 expands the buffycoat 902 between the main body of the float and the inner wall of theprimary vessel. The buffy coat 902 can be trapped within an area betweenthe float 804 and the primary vessel 802.

FIGS. 10A and 10B show a first seal being formed to prevent fluids frommoving up or down within the primary vessel. For convenience, FIG. 10Ashall be used to describe the method, though the method applies equallyto FIG. 10B. The first seal also inhibits float movement. The firstsealing ring 600 may be placed at approximately a lower end of the mainbody of the float 804. The first sealing ring 600 exerts circumferentialor radial forces on the primary vessel 802, thereby causing the primaryvessel 802 to collapse inwardly against the float 804. Magnified view1102 shows the first sealing ring 600 tightened around the float andprimary vessel system 800. The first sealing ring 600, having beenplaced at an interface of the buffy coat 902 and the red blood cells903, causes the primary vessel 802 to collapse inwardly until a seal isformed between the primary vessel 802 and the float 804. An outer wallof the first sealing ring 600 may sit flush with an outer wall of theprimary vessel 802; the outer wall of the first sealing ring 600 mayextend past the outer wall of the primary vessel 802; or, the outer wallof the primary vessel 802 may extend past the outer wall of the firstsealing ring 600. The first sealing ring 600 remains tightened tomaintain the seal, which prevents fluids from moving past the seal inany direction. The first sealing ring 600 may also remain in tension.Alternatively, the first sealing ring 600 may be overtightened and thenthe force applied to the first sealing ring 600 is removed. The firstsealing ring 600 may expand slightly, though still remains constricted.

To apply the first sealing ring 600 and thereby form the seal, a clampmay be used to circumferentially apply a force directed toward thecentral axis of the primary vessel 802 to the first sealing ring 600 andthe float and primary vessel system 800. The first sealing ring 600 isplaced around the float and primary vessel system 800 after the floatand primary vessel system 800 have undergone density-based separation,such as by centrifugation. The first sealing ring 600 and float andprimary vessel system 800 are then placed into the clamp. The clamp mayinclude a shelf to support the first sealing ring 600 against theprimary vessel 802. Operation of the clamp may be automated or may beperformed manually. Alternatively, the clamp may form a seal between thefloat 804 and primary vessel 802 without the inclusion of the firstsealing ring 600. Alternatively, a seal may be formed between the float804 and the primary vessel 802 such as by ultrasonic welding; or byapplying heat or a temperature gradient to deform and/or melt theprimary vessel 802 to the float 804. For the sake of convenience, themethods are described with reference to the seal between the float andthe primary vessel, but the methods described below are not intended tobe so limited in their application and may be performed without theseal.

When operation of the clamp is automated, a motor causes translation ofeither a collet, including collet fingers, or a pressure member to causecompression of the collet fingers. The motor may be connected to thecollet or the pressure member by a shaft, such as a cam shaft, and oneor more gears. A base engages and holds the object. When the collet isdriven by the motor, the pressure member remains stationary. When thepressure member is driven by the motor, the collet remains stationary.The clamp may include a release, so as to cause the pressure member toslide off of the collet fingers 804, thereby removing the clampingforce.

Alternatively, the clamp may be, but is not limited to, a collet clamp,an O-ring, a pipe clamp, a hose clamp, a spring clamp, a strap clamp, ora tie, such as a zip tie. The clamp may be used without a first sealingring to provide a seal between a float and a tube.

The plasma 901 may be removed from the primary vessel 802, as shown inFIG. 11A, such as by pipetting, suctioning, pouring, or the like.Returning to FIG. 7, in block 706, a clearing fluid 1102 may be added tothe primary vessel 802 and the system undergoes centrifugation again, asshown in FIGS. 11B-11C. The clearing fluid 1102 has a density greaterthan the buffy coat 902, but may be layered on top of the buffy coat902. It may be desirous to gently layer the clearing fluid 1102 on topof the buffy coat 902 to inhibit mixing of the clearing fluid 1102 withthe buffy coat 902. During centrifugation, the clearing fluid 1102 movesunderneath the buffy coat 902 but remains above the first sealing ring600. After centrifugation, the buffy coat 902, having a density lessthan the clearing fluid 1102, rests on top of the clearing fluid 1102.An appropriate amount of the clearing fluid 1102, such as up to 500milliliters, may be added to the primary vessel 802, such that the buffycoat 902 is not between the main body of the float 804 and the innerwall of the primary vessel 802. The clearing fluid 1102 may be miscibleor immiscible with the suspension fluid and inert with respect to thesuspension materials. Examples of suitable clearing fluids include, butare not limited to, solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), an organic solvent, a liquid wax, anoil, a gas, and combinations thereof; olive oil, mineral oil, siliconeoil, immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, and combinations thereof; ionic liquids;polymer-based solutions; surfactants; perfluoroketones, such asperfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones,hydrofluoroethers, hydro fluorocarbons, perfluorocarbons,perfluoropolyethers, silicon and silicon-based liquids, such asphenylmethyl siloxane; and combinations thereof.

A second sealing ring 600 may be placed at approximately an upper end ofthe main body of the float 804, as shown in FIG. 11D. An antibodycocktail may be added to the primary vessel 802. The antibody cocktailmay include at least one antibody to bind to a receptor on a targetmaterial. The at least one antibody of the antibody cocktail may beconjugated to a first complementary molecule, which may bind to a secondcomplementary molecule. The selected antibodies may bind to the antigensincluding, but not limited to, EGFR, HER-2, HER-3, HER-4, VE-cadherin,CD144, CD147, EPCAM, PSMA, PSA, CD271, MUC1, SLUG, SNAIL, TWIST,E-cadherin, N-cadherin, CD71, and, CD146. Alternatively, a plurality ofantibodies may be added to the sample to bind to at least one targetmaterial.

Returning to FIG. 7, in block 708, an enrichment agent may be added tothe primary vessel 802 to select or aid in selecting the target materialfrom amongst the remainder of the sample. The enrichment agent may altera physical characteristic of the target material (i.e. density, abilityto respond to a magnetic gradient, or the like). For example, the targetmaterial may be circulating tumor cells or fetal cells. The enrichmentagent may be, for example, immunomagnetic beads, buoyant beads,high-density beads, chemicals to change the density of the targetmaterial, or the like. It should be noted, however, that the enrichmentagent does include any fluorescent molecules configured to respond anexcitation light, such as one used in flow cytometry or fluorescentmicroscopy. Examples of suitable enrichment agents include solutionssuch as, a solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), a complex, branch glucan (e.g.Dextran), cesium chloride, sucrose, sugar-based solutions, polymersolutions, multi-phase polymer solutions, tetrameric antibody complexes(e.g. RosetteSep) or the like; or particles, such as beads (composed ofat least one of a metal, silica, glass, a polymer, or the like),nanoparticles, metal-based compounds, metal complexes, lipids, sugars,or the like. The enrichment agent, such as the particles, nanoparticles,complexes, or compounds, may be conjugated to the second complementarymolecule, which may bind to the first complementary molecule of the atleast one antibody of the antibody cocktail. Alternatively, theenrichment agent, such as the particles, nanoparticles, complexes, orcompounds, may be conjugated to the at least one antibody of theantibody cocktail, such that the enrichment agent may bind directly tothe target material. Furthermore, a plurality of enrichment agents maybe used. For example, immunomagnetic beads may be used and buoyant glassbeads. The buoyant glass beads may be used to alter the density of thetarget material and the immunomagnetic beads may be used, in conjunctionwith a magnet, to further enrich the target material. The particles maybe approximately 0.1-5.0 μm in size.

These first and second complementary molecules may bind to each other bycovalent, ionic, dipole-dipole interactions, London dispersion forces,Van der Waal's forces, hydrogen bonding, or other chemical bonds. Thefirst complementary molecule, whether introduced to the particle throughbinding, coating, or attaching, may include, but is not limited to, anavidin, such as streptavidin or neutravidin; Protein A, Protein G,Protein L; biotin; a biotin analog; an aptamer; a primary antibody thatbinds to biomarkers, including but not limited to, EpCAM, AMACR,Androgen receptor, CD146, CD227, CD235, CD24, CD30, CD44, CD45, CD56,CD71, CD105, CD324, CD325, MUC1, CEA, cMET, EGFR, Folate receptor, HER2,Mammaglobin, or PSMA; a ligand, such as EGF, HGF, TGFα, TGFβ superfamilyof ligands, IGF1, IGF2, Wnt signaling proteins, FGF signaling ligands,amphiregulin, HB-EGF, neuregulin signaling ligands, MSP, VEGF family ofligands, betacellulin, epiregulin, epigen, hedgehog signaling ligands;IgG, IgM; scFv, Fab, sdAb; an antibody-like molecule that binds to abiomarker; or a second antibody.

The second complementary molecule may include, but is not limited to, anavidin, such as streptavidin or neutravidin; Protein A, Protein G,Protein L; biotin; a biotin analog; an aptamer; a primary antibody thatbinds to biomarkers, including but not limited to, EpCAM, AMACR,Androgen receptor, CD146, CD227, CD235, CD24, CD30, CD44, CD45, CD56,CD71, CD105, CD324, CD325, MUC1, CEA, cMET, EGFR, Folate receptor, HER2,Mammaglobin, or PSMA; a ligand, such as EGF, HGF, TGFα, TGFβ superfamilyof ligands, IGF1, IGF2, Wnt signaling proteins, FGF signaling ligands,amphiregulin, HB-EGF, neuregulin signaling ligands, MSP, VEGF family ofligands, betacellulin, epiregulin, epigen, hedgehog signaling ligands;IgG, IgM; scFv, Fab, sdAb; an antibody-like molecule that binds to abiomarker; or a second antibody.

The enrichment agent and the depletion agent may be added simultaneouslyor at different points during the process.

In block 710, a collector, such as the collector 200, may be insertedinto the primary vessel 802. In block 712, a processing vessel 402including the collection fluid 412 is inserted into the collector 200.FIG. 11D shows the collector 200 inserted into the primary vessel 802and also forming a seal 1106 between the second end 208 of the collector200 and the inner wall of the primary vessel 802.

Alternatively, after inserting the collector, 200, a layering fluid maybe added to the primary vessel 802 through the collector 200, such as bya fluid layering device, to fill the volume of the primary vesselbeneath the collector 200. The fluid layering device may include a motorconnected to a rod which is also connected to a piston. The fluidlayering device may also include a switch to activate or de-activate themotor. When the motor is activated, the rod, which may be threaded, mayrotate, thereby causing the piston to move up and down, thereby creatinga pressure gradient to expel a layering fluid from the fluid layeringdevice, including a plug, when the fluid layering device is insertedinto the collector 200, such that the cannula 216 extends through theplug. Alternatively, the rod may move up and down, thereby causing thepiston to move up and down, hence creating the pressure gradient.Alternatively, a cam mechanism, servomotor, diaphragm, or rack andpinion system may be used to either move the piston up and down tocreate the pressure gradient or to create the pressure gradient withoutthe piston. Alternatively, the fluid layering device may be a syringe.

The layering fluid has a greater density than the density of the targetmaterial of the suspension and the collection fluid. The collectionfluid 412 may be miscible or immiscible in the suspension fluid.Examples of suitable collection fluids include, but are not limited to,solution of colloidal silica particles coated with polyvinylpyrrolidone(e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g.OptiPrep), a liquid wax, an oil, a gas, and combinations thereof; oliveoil, mineral oil, silicone oil, immersion oil, mineral oil, paraffinoil, silicon oil, fluorosilicone, perfluorodecalin,perfluoroperhydrophenanthrene, perfluorooctylbromide, and combinationsthereat ionic liquids; polymer-based solutions; surfactants;perfluoroketones, such as perfluorocyclopentanone andperfluorocyclohexanone, fluorinated ketones, hydrofluoroethers,hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon andsilicon-based liquids, such as phenylmethyl siloxane; and combinationsthereof.

Returning to FIG. 7, in block 714, the primary vessel, the collector,and the processing vessel are centrifuged. During centrifugation, thecollection fluid 412 moves from the processing vessel 402 into theprimary vessel 802 via the cannula 216 of the collector 200 anddisplaces the buffy coat 902 from the primary vessel 802 into theprocessing vessel 402 via cannula 216 the collector 200, as shown inFIG. 11E. The processing vessel 402, now including the buffy coat 902,may be removed from the collector 200, as shown in FIG. 11F.

The collection fluid may be miscible or immiscible with the suspensionfluid and inert with respect to the suspension materials. The collectionfluid 412 has a greater density than the density of the target materialof the suspension (the density may be less than the density of at leastone other suspension fraction or the density may be greater than all ofthe suspension fractions) and is inert with respect to the suspensionmaterials. Examples of suitable collection fluids include, but are notlimited to, solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), a complex, branch glucan (e.g.Dextran), cesium chloride, sucrose, sugar-based solutions, polymer-basedsolutions, surfactants, an organic solvent, a liquid wax, an oil, oliveoil, mineral oil, silicone oil, and ionic liquids; perfluoroketones,such as perfluorocyclopentanone and perfluorocyclohexanone, fluorinatedketones, hydrofluoroethers, hydrofluorocarbons, perfluorocarbons,perfluoropolyethers, silicon and silicon-based liquids, such asphenylmethyl siloxane.

The processing vessel 402 may also include a processing solution toeffect a transformation on the target material when the target materialenters the processing vessel 402. The processing solution may be apreservative, a fixative, a cell adhesion solution, a dye, a freezingstabilization media, or the like. Unlike the collection fluid, most, ifnot all, of the processing solution remains within the processingreceptacle 402 upon centrifugation, thereby effecting the transformationon the target material in one manner or another (i.e. preserving,fixing, increasing adhesion properties, or the like) in the processingvessel 402. The processing solution may be introduced as a liquid or asa liquid container in a casing. The casing may be dissolvable in anaqueous solution but not in the collection fluid (such as a gel cap);or, the casing may be breakable, such that the casing breaks when theprocessing vessel 402 is shaken in a vortex mixer. Additionally, morethan one processing solution may be used.

Furthermore, when the vessel includes a septum in the closed end, theplasma, for example, may be removed through the septum with a needle,syringe, by draining, or the like. The plasma may then be fartherprocessed and analyzed.

Sequential density fractionation is the division of a sample intofractions or of a fraction of a sample into sub-fractions by a step-wiseor sequential process, such that each step or sequence results in thecollection or separation of a different fraction or sub-fraction fromthe preceding and successive steps or sequences. In other words,sequential density fractionation provides individual sub-populations ofa population or individual sub-sub-populations of a sub-population of apopulation through a series of steps. For example, buffy coat is afraction of a whole blood sample. The buffy coat fraction may be furtherbroken down into sub-fractions including, but not limited to,reticulocytes, granulocytes, lymphocytes/monocytes, and platelets. Thesesub-fractions may be obtained individually by performing sequentialdensity fractionation.

FIG. 12 shows an example method 1200 for retrieving a target materialusing sequential density fractionation. The example method 1200 issimilar to the example method 700, except the example method 1200collects the target material by sequential density fractionation. Afterthe steps of block A have been performed, as shown in FIG. 7, sequentialdensity fractionation is performed, as seen in block 1202. Block 1202 isalso a snapshot of the sequential density fractionation steps. In block1204, an n^(th) processing receptacle including an n^(th) collectionfluid is inserted into the collector, such that n^(th) is greater thanor equal to first (i.e. second, third, fourth, and so on). In block1206, the system is centrifuged to collect a fraction or sub-fractionand the nth processing receptacle is removed. In block 1208, theoperator determines whether or not the desired fraction or sub-fractionis obtained. When the desired fraction or sub-fraction is obtained, theprocess may stop as shown in block 1210, though the process may continueuntil all fractions or sub-fractions are obtained. When the desiredfraction or sub-fractions is not yet obtained, the process restarts atblock 1204. The processing receptacles may also include a processingsolution to effect a change on the respective sub-fractions. Two or moreprocessing receptacles and respective collection fluids may be useddepending on the number of fractions or sub-fractions desired forseparation and collection. Each successive collection fluid is denserthan the preceding collection fluid. Similarly, each successive fractionor sub-fraction is denser than the preceding fraction or sub-fraction.Once collected, the consecutive sub-fractions may be analyzed, such asfor diagnostic, prognostic, research purposes, to determine componentscharacteristics (i.e. a complete blood count), how those characteristicschange over time, or the like.

The target material may be spread onto slides or retained in an aqueoussuspension. The target material may be analyzed using any appropriateanalysis method or technique, though more specifically extracellular andintracellular analysis including intracellular protein labeling;chromogenic staining; molecular analysis; genomic analysis or nucleicacid analysis, including, but not limited to, genomic sequencing, DNAarrays, expression arrays, protein arrays, and DNA hybridization arrays;in situ hybridization (“ISH”—a tool for analyzing DNA and/or RNA, suchas gene copy number changes); polymerase chain reaction (“PCR”); reversetranscription PCR; or branched DNA (“bDNA”—a tool for analyzing DNAand/or RNA, such as mRNA expression levels) analysis. These techniquesmay require fixation, permeabilization, and/or isolation (such as bypicking) of the target material prior to analysis. Some of theintracellular proteins which may be labeled include, but are not limitedto, cytokeratin (“CK”), actin, Arp2/3, coronin, dystrophin, FtsZ,myosin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1,Akt2, c-myc, caspases, survivin, p27^(kip), FOXC2, BRAF, Phospho-Akt1and 2, Phospho-Erk1/2, Erk1/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK,KRAS, ALKH1, Twist1, Snail1, ZEB1, Fibronectin, Slug, Ki-67, M30,MAGEA3, phosphorylated receptor kinases, modified histones,chromatin-associated proteins, and MAGE. To fix, permeabilize, or label,fixing agents (such as formaldehyde, formalin, methanol, acetone,paraformaldehyde, or glutaraldehyde), detergents (such as saponin,polyoxyethylene, digitonin, octyl β-glucoside, octyl β-thioglucoside,1-S-octyl-β-D-thioglucopyranoside, polysorbate-20, CHAPS, CHAPSO,(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol or octylphenolethylene oxide), or labeling agents (such as fluorescently-labeledantibodies, enzyme-conjugated antibodies, Pap stain, Giemsa stain, orhematoxylin and eosin stain) may be used.

After collection, the target material may also be imaged or may undergoflow cytometry. To be imaged, a solution containing a fluorescent probemay be used to label the target material, thereby providing afluorescent signal for identification and characterization, such asthrough imaging. The fluorescent probe may be added to the primaryvessel after the second sealing ring has been applied or after at leastone non-target material, such as the plasma, has been removed. Thesolution containing the fluorescent probe may be added to the suspensionbefore the suspension is added to the vessel, after the suspension isadded to the vessel but before centrifugation, or after the suspensionhas undergone centrifugation. The fluorescent probe includes afluorescent molecule bound to a ligand. The target material may have anumber of different types of surface, intracellular, or nuclear markers.Each type of surface marker is a molecule, such an antigen, capable ofattaching a particular ligand, such as an antibody. As a result, ligandsmay be used to classify the target material and determine the specifictype of target materials present in the suspension by conjugatingligands that attach to particular surface markers with a particularfluorescent molecule. Examples of suitable fluorescent moleculesinclude, but are not limited to, quantum dots; commercially availabledyes, such as fluorescein, Hoechst, FITC (“fluorescein isothiocyanate”),R-phycoerythrin (“PE”), Texas Red, allophycocyanin, Cy5, Cy7, cascadeblue, DAPI (“4′,6-diamidino-2-phenylindole”) and TRITC(“tetramethylrhodamine isothiocyanate”); combinations of dyes, such asCY5PE, CY7APC, and CY7PE; and synthesized molecules, such asself-assembling nucleic acid structures. Many solutions may be used,such that each solution includes a different type of fluorescentmolecule bound to a different ligand.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents:

We claim:
 1. A method comprising the steps of: providing a primaryvessel comprising an open end and a suspension comprising a targetmaterial; adding a positive enrichment reagent to the suspension forpositive selection of the target material; inserting a device comprisinga cannula into the open end of the primary vessel, wherein the deviceextends upwardly from the open end of the primary vessel; providing aprocessing vessel comprising a first end comprising a resealable plug,and an inner cavity; mating the processing vessel with the device,wherein the cannula extends through the resealable plug of theprocessing vessel and accesses the inner cavity of processing vessel,and wherein the processing vessel extends upwardly from the device;adding a displacement fluid comprising a density greater than a densityof the target material to the inner cavity of the processing vessel; andcentrifuging the primary vessel, the device, the displacement fluid, andthe processing vessel, wherein during centrifuging, the displacementfluid flows into the primary vessel via the device and displaces thetarget material from the primary vessel, which flows into the processingvessel through the device via the cannula.
 2. The method of claim 1,further comprising the step of: separating the suspension into fractionsprior to inserting step.
 3. The method of claim 2, further comprisingthe step of: adding a first depletion agent to the suspension to removea first sample fraction from the suspension or to change the density ofat least a first non-target material relative to the density of thetarget material.
 4. The method of claim 3, further comprising the stepof: adding a second depletion agent to the suspension to remove a secondsample fraction from the suspension or to change the density of at leasta second non-target material relative to the density of the targetmaterial.
 5. The method of claim 2, further comprising the step of:removing at least a portion of a non-target material from the primaryvessel after the separating step and before the inserting step.
 6. Themethod of claim 5, further comprising the step of: adding a clearingfluid to the primary vessel after the removing step, the clearing fluidhaving a density greater than at least the target material.
 7. Themethod of claim 6, further comprising the step of: adding a float to theprimary vessel prior to the separating step.
 8. The method of claim 7,further comprising the step of: forming a first seal between the floatand the primary vessel by clamping after the separating step, theremoving step, or adding the clearing fluid.
 9. The method of claim 8,further comprising the step of: centrifuging the primary vessel afterforming the first seal.
 10. The method of claim 9, further comprisingthe step of: forming a second seal between the float and the primaryvessel by clamping after the centrifuging step performed after formingthe first seal, wherein the first seal is formed between a lower end ofa main body of the float and an inner wall of the primary vessel, andwherein the second seal is formed between an upper end of the main bodyof the float and the inner wall of the primary vessel.
 11. The method ofclaim 1, further comprising the step of: forming a seal between an endof the device and an inner wall of the primary vessel to maintain afluid-tight sealing engagement between the end of the device and innerwall of the primary vessel.
 12. The method of claim 1, wherein thedisplacement fluid is selected from the group consisting of: a solutionof colloidal silica particles coated with polyvinylpyrrolidone, apolysaccharide solution, iodixanol, a liquid wax, an oil, a gas, oliveoil, mineral oil, silicone oil, immersion oil, mineral oil, paraffinoil, silicon oil, fluorosilicone, perfluorodecalin,perfluoroperhydrophenanthrene, perfluorooctylbromide, ionic liquids, apolymer-based solution, a surfactant, a perfluoroketone,perfluorocyclopentanone, perfluorocyclohexanone, a fluorinated ketone, ahydrofluoroether, a hydrofluorocarbon, a perfluorocarbon, aperfluoropolyether, silicon, a silicon-based liquid, phenylmethylsiloxane, and combinations thereof.
 13. The method of claim 1, whereinthe positive enrichment reagent is a solution of colloidal silicaparticles coated with polyvinylpyrrolidone, a polysaccharide solution,iodixanol, a complex, branch glucan, cesium chloride, sucrose, asugar-based solution, a polymer solution, a multi-phase polymersolution, a tetrameric antibody complex, a particle, a nanoparticle, ametal-based compound, a metal complex, a lipid, or a sugar.
 14. A systemcomprising: a primary vessel comprising an open end, a suspensioncomprising a target material, and a positive enrichment reagent to thesuspension for positive selection of the target material; a processingvessel comprising a first end comprising a resealable plug, an innercavity, and a displacement fluid comprising a density greater than adensity of the target material, wherein the displacement fluid islocated within the inner cavity; a device at least partially locatedwithin the open end of the primary vessel, the device comprising acannula, wherein the cannula extends through the resealable plug of theprocessing vessel and accesses the inner cavity of the processingvessel, thereby mating the processing vessel and the device and fluidlyconnecting the primary vessel to the processing vessel, wherein thedevice extends upwardly from the open end of the primary vessel, andwherein the processing vessel extends upwardly from the device.
 15. Thesystem of claim 14, the primary vessel further comprising a clearingfluid comprising a density greater than a density of at least the targetmaterial.
 16. The system of claim 14, further comprising: a floatlocated at a longitudinal position within the primary vessel.
 17. Thesystem of claim 16, further comprising a sealing ring locatedcircumferentially around the primary vessel at the same longitudinalposition as at least a portion of the float within the primary vessel.18. The system of claim 14, further comprising a seal between an end ofthe device and an inner wall of the primary vessel to maintain afluid-tight sealing engagement between the end of the device and innerwall of the primary vessel.
 19. The system of claim 14, wherein thedisplacement fluid is selected from the group consisting of: a solutionof colloidal silica particles coated with polyvinylpyrrolidone, apolysaccharide solution, iodixanol, a liquid wax, an oil, a gas, oliveoil, mineral oil, silicone oil, immersion oil, mineral oil, paraffinoil, silicon oil, fluorosilicone, perfluorodecalin,perfluoroperhydrophenanthrene, perfluorooctylbromide, ionic liquids, apolymer-based solution, a surfactant, a perfluoroketone,perfluorocyclopentanone, perfluorocyclohexanone, a fluorinated ketone, ahydrofluoroether, a hydrofluorocarbon, a perfluorocarbon, aperfluoropolyether, silicon, a silicon-based liquid, phenylmethylsiloxane, and combinations thereof.